Expression Analysis Of Proteins Encoded By Genome Segment 3 And Segment 9 From Rice Black Streaked Dwarf Virus

The full-length cDNA of rice black-streaked dwarf virus (RBSDV) genomic segment 9 (S9) of an isolate (Hbm) causing maize rough dwarf disease in Hubei Province was cloned by RT-PCR and its sequence was determined. The Hbm-S9 consisted of 1900 base pairs, and contained two non-overlapping ORFs. ORFl(1043nt) encoded a 40 kDa protein(P9-l) which consisted of 347 amino acids , and ORF2(682nt) encoded a 24 kDa protein (P9-2) which consisted of 209 amino acids. Sequence analysis showed that Hbm-S9 shared 94.4-98.9 % and 96.0-99.0 % identities at nucleotide sequence and amino acid level respectively with different Chinese isolates of RBSDV. Furthermore, the sequences identities to the S9 of a Japanese isolate reported were higher than to the S8 of maize rough dwarf virus isolated and reported in Italy. These results further supported that maize rough dwarf disease was caused by RBSDV in China. The proteins encoded by segment 9 were expressed as fusion proteins in Escherichia coli at high level. Western blot revealed that an antibody against the expressed Hbm S9 ORF1 product could detect a single protein of the predicted size in total proteins extracted from Hbm infected maize leaves.Two proteins encoded by Hbm-S3 5' and 3' terminal sequences respectively were expressed in E. coli using pET expression vectors and purified. Antibodies raised against the expressed products were used as probes to analysis the propertites of the protein encoded by Hbm-S3.Western blots showed that antisera to the Hbm-P3-N and Hbm-P3-C each detected a protein of the predicted size in total proteins extracted from maize leaves infected by RBSDV(Hbm) while antibody to the expressed Hbm-S3-3' product reacted with a 130 kDa protein ectracted from purifed virus particles. These results seem to suggest that the protein encoded by RBSDV (Hbm)- S3 was one of the structural proteins.